The toxic mechanism of herbicide butachlor to induce extremely high lethality in marine flatfish flounder, Paralichthys Olivaceus, was analyzed by histopathological examination, antioxidant enzymes activities and ATP content assay. Histopathological examination of gill, liver and kidney of exposed fishes showed that gill was a target organ of butachlor. The butachlor seriously impaired the respiration of gills by a series of lesions such as edema, lifting and detachment of lamellar epithelium, breakdown of pillar cells, and blood congestion. The dysfunction of gill respiration caused suffocation to the exposed flounder with extremely high acute lethality. Antioxidant enzyme activity assay of the in vitro cultured flounder gill (FG) cells exposed to butachlor indicated that butachlor markedly inhibited the antioxidant enzyme activities of Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). Furthermore, along with the decline of antioxidant enzyme activities, ATP content in the exposed FG cells decreased, too. This infers that the oxidative stress induced by butachlor can inhibit the production of cellular ATP. Similar decrease of ATP content was also observed in the exposed flounder gill tissues. Taken together, as in FG cells, butachlor possibly induced a short supply of ATP in pillar cells by inhibiting the antioxidant enzyme activities and then affecting the contractibility of the pillar cells, which in turn resulted in the blood congestion and suffocation of exposed flounder.
Edwardsiella piscicida is an intracellular pathogenic bacterium accounting for significant losses in farmed fish. Currently, cellular and molecular mechanisms underlying E. piscicida–host cross-talk remain obscure. In this study, we revealed that E. piscicida could increase microtubule-associated protein L chain 3 (LC3) puncta in grass carp (Ctenopharyngodon idella) monocytes/macrophages and a carp cell line, Epithelioma papulosum cyprini. The autophagic response was confirmed by detecting the colocalization of E. piscicida with LC3-positive autophagosomes and LysoTracker-probed lysosomes in the cells. Moreover, we unveiled the autophagic machinery targeting E. piscicida by which the nucleotide-binding oligomerization domain receptor 1 (NOD1) functioned as an intracellular sensor to interact and recruit autophagy-related gene (ATG) 16L1 to the bacteria. Meanwhile, E. piscicida decreased the mRNA and protein levels of NOD1 and ATG16L1 in an estrogen-related receptor-α–dependent manner, suggesting a possible mechanism for this bacterium escaping autophagy. Subsequently, we examined the effects of various E. piscicida virulence factors on NOD1 expression and found that two of them, EVPC and ESCB, could reduce NOD1 protein expression via ubiquitin-dependent proteasomal degradation. Furthermore, an intrinsic regulator IFN-γ was found to enhance the colocalization of E. piscicida with NOD1 or autophagosomes, suggesting its involvement in the interaction between autophagy and E. piscicida. Along this line, a short-time treatment of IFN-γ caused intracellular E. piscicida clearance through an autophagy-dependent mechanism. Collectively, our works demonstrated NOD1-mediated autophagy–E. piscicida dialogues and uncovered the molecular mechanism involving autophagy against intracellular bacteria in fish.
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